The long-term objectives of the proposed research are to understand how specificity is determined within a family of eukaryotic regulatory transcription factors. The ets gene family, which is found throughout the metazoa, has 27 human paralogs. ETS proteins have highly conserved DNA binding properties, yet display unique distinct biological properties. Thus, specificity must have evolved to direct these proteins to function in unique biological contexts. This proposal focuses on how signaling pathways affect the function of ETS proteins to give distinct activities. Aim I investigates the autoinhibition of Ets-1 DNA binding, including reinforcement by calciumdependent phosphorylation. Biochemical and biophysical approaches will test the role of protein dynamics and conformational change in the regulation of Ets-1 DNA binding. A phosphorylation-dependent rheostat model of Ets-1 regulation will be tested in vitro and in cells. Aim II investigates Ets-1 and highly-related Ets-2 as effectors of Ras/MAPKinase signaling. We will focus on the macromolecular interfaces that mediate the docking of the MAPK ERK2 to Ets-1 and Ets-2 and the phosphorylation-dependent recruitment of the co-activators CBP/p300. In addition, two recently discovered Ets-1 modifications, acetylation and sumolation, will be investigated. Aim III will identify authentic transcriptional targets for Ets-1 and Ets-2 to investigate how signaling impinges on the activity of Ets-1 and Ets-2 at genomic loci. We will test the role of post-translational modifications and factor assembly in gene activation and repression. Ras/MAPK signaling and the ETS family of transcription factors regulate genes necessary for control of the cell cycle, apoptosis, and normal differentiation. Mutation of human ras and ets genes contribute to cellular dysfunction in cancer. Indeed, the ras gene is mutated in 20% of human cancers. Therefore, deciphering the molecular function of Ets-1 and Ets-2 within Ras-dependent signal transduction will provide new insight and guide development of new tools to confront the cancer problem.